肠杆菌科细菌对多粘菌素获得性耐药的机制研究

Enterobacter cloacae is a common bacterial pathogen causing human infections. Polymyxin is the last resort of antimicrobial agents against it. However, E. cloacae strains with acquired resistance to polymyxin have emerged. The mechanism mediating acquired resistance to polymyxin in E. cloacae remains largely uninvestigated. In this project, 5 polymyxin-resistant clinical isolates of E. cloacae will be subjected to whole genomic sequencing and comparative genomics, two-way shot-gun cloning (cloning DNA from polymyxin-resistant strains into susceptible ones and vice versa), targeted cloning of certain genes, site-directed mutagenesis, and transcriptome sequencing and analysis. These methods will in tandem reveal the mechanism mediating acquired resistance to polymyxin in these isolates. In addition, one polymyxin-susceptible E. cloacae strain will be included into in vitro selection for polymyxin-resistant mutants and Tn5 transposon insertion experiments to identify candidate genes mediating acquired polymyxin resistance in laboratory conditions. The candidate genes mediating resistance to polymyxin will be confirmed or rejected through complementation experiments using wild-type alleles. The investigation on wild-type clinical isolates will reveal polymyxin resistance mechanism occurred in nature, while the study using in vitro selection and Tn5 insertion for polymyxin-susceptible isolates will shed light on resistance mechanisms that may not have been identified in wild isolates but will possibly occur in nature. The combination of the two aspects will help us comprehensively appreciate acquired mechanisms mediating polymyxin resistance in E. cloacae. The generated data and information will be critical to design schemes for rapid identification of polymyxin-resistant isolates and therefore may assist the rational clinical use of antimicrobial agents and the investigation for the prevention and control of healthcare-associated infections. New targets for drug development may be also inferred from such data.

阴沟肠杆菌科细菌是临床常见病原菌，多粘菌素是治疗其感染的最后底线。对多粘菌素获得性耐药的阴沟肠杆菌科菌株已涌现，但其耐药机制尚未被阐明。本项目将首先对5株多粘菌素耐药阴沟肠杆菌临床菌株，通过全基因组测序比较、双向鸟枪克隆、基因的靶向克隆、定点突变和转录组测序等方法，对多粘菌素的获得性耐药机制进行抽丝剥茧式解析。还将选取阴沟肠杆菌敏感株1株，通过体外诱导耐药和Tn5转座子插入试验建立对多粘菌素耐药的变异株；并通过基因组测序和反向PCR等揭示介导多粘菌素耐药的基因。对临床株的研究将揭示在自然界中已发生的耐药机制；而对敏感株在实验条件下诱导耐药进行研究将阐释可能尚未在临床株中发现的耐药机制。将两者结合，将揭示阴沟肠杆菌对多粘菌素获得性耐药的机制，所获信息将有助于开发耐药性快速检测方法以协助抗菌药物使用和医院感染流行病学调查等，并为药物开发寻找靶位。

肠杆菌科细菌是临床最常见病原菌，多粘菌素是治疗其感染的最后底线。对多粘菌素获得性耐药的肠杆菌科菌株已涌现，本项目对92株阴沟肠杆菌复合体菌株进行检测，发现一半对多粘菌素耐药。我们对其中一株对多粘菌素高度耐药的菌株开展了深入研究，发现了其对多粘菌素耐药的全新机制。通过Tn5转座子插入突变和定点敲除，发现除了已知的PhoP双组分调节系统之外，还有跨膜蛋白编码基因dedA和外排泵基因tolC也是多粘菌素耐药所必需。虽然，这些基因被插入或敲除而失活时，菌株变为多粘菌素敏感；但补回多粘菌素敏感菌株的这些基因也能恢复这些插入或敲除株对多粘菌素耐药，提示其多粘菌素耐药另有机制。我们接着通过鸟枪克隆，发现一个编码72个氨基酸的基因介导了该菌株对多粘菌素的高度耐药，是一个全新的多粘菌素耐药机制。通过预测发现该基因编码跨膜蛋白；通过表达水平研究发现其很可能作用于PhoP-PhoQ双组分和AcrAB-TolC外排泵两个系统而介导了对多粘菌素的高水平耐药。此外，我们还对医院污水和临床标本分离的耐多粘菌素的肠杆菌科菌株进行了质粒介导的多粘菌素耐药基因的研究。发现了多个菌种（大肠埃希菌、肺炎克雷伯菌和克吕沃尔菌）携带mcr-1基因，而携带mcr-1的大肠埃希菌属于多个ST类型、mcr-1在这些菌株中由多种不同的质粒（IncX4、IncI2、IncHI2、IncP和噬菌体类似）携带、而且位于多种不同的基因环境中。IncX4和IncI2是最常见的携带mcr-1的质粒，其中通过进化分析我们发现IncX4质粒很可能是获得mcr-1后再逐渐分化，而IncI2质粒则是多次获得mcr-1。我们发现位于mcr-1上游的插入序列ISApl1参与了该基因的移动。同时，我们还发现了两个新的mcr-1的亚型，分别是mcr-1.4和mcr-1.7。我们还在大肠埃希菌中发现了另一个质粒介导的多粘菌素耐药基因mcr-3，为一种新的亚型mcr-3.5。mcr-3由TnAs3携带。以上所获信息将有助于开发耐药性快速检测方法以协助抗菌药物使用和医院感染流行病学调查等，并为药物开发寻找靶位。